Amine Oxidation Mediated by Lysine-Specific Demethylase 1: Quantum Mechanics/Molecular Mechanics Insights into Mechanism and Role of Lysine 661

Abstract

We report classical molecular dynamics (MD) simulations and combined quantum mechanics/molecular mechanics (QM/MM) calculations to elucidate the catalytic mechanism of the rate-determining amine oxidation step in the lysine-specific demethylase 1 (LSD1)-catalyzed demethylation of the histone tail lysine (H3K4), with flavin adenine dinucleotide (FAD) acting as cofactor. The oxidation of substrate lysine (sLys) involves the cleavage of an α-CH bond accompanied by the transfer of a hydride ion equivalent to FAD, leading to an imine intermediate. This hydride transfer pathway is shown to be clearly favored for sLys oxidation over other proposed mechanisms, including the radical (or single-electron transfer) route as well as carbanion and polar-nucleophilic mechanisms. MD simulations on six NVT ensembles (covering different protonation states of sLys and K661 as well as the K661M mutant) identify two possible orientations of the reacting sLys and FAD subunits (called "downward" and "upward"). Calculations at the QM(B3LYP-D/6-31G*)/CHARMM22 level provide molecular-level insights into the mechanism, helping to understand how LSD1 achieves the activation of the rather inert methyl-CH bond in a metal-free environment. Factors such as proper alignment of sLys (downward orientation), transition-state stabilization (due to the protein environment and favorable orbital interactions), and product stabilization via adduct formation are found to be crucial for facilitating the oxidative α-CH bond cleavage. The current study also sheds light on the role of important active-site residues (Y761, K661, and W695) and of the conserved water-bridge motif. The steric influence of Y761 helps to position the reaction partners properly, K661 is predicted to get deprotonated prior to substrate binding and to act as an active-site base that accepts a proton from sLys to enable the subsequent amine oxidation, and the water bridge that is stabilized by K661 and W695 mediates this proton transfer.

13C kinetic isotope effects on the reaction of a flavin amine oxidase determined from whole molecule isotope effects

“…For example, Skarpeli-Liati et al [39] have reported an average 15 N isotope effect of 0.998 ± 0.004 for the one electron oxidation of N-methyl- and N, N-dimethylbenzylamines. This is consistent with calculated values of 0.996–0.998 for the formation of the nitrogen radical [8, 40]. In contrast, the average of reported 15 (k cat /K m ) values for flavin amine oxidases is 0.992 ± 0.005 [40–43], in good agreement with the value of 0.992–0.993 calculated for a hydride transfer mechanism [8, 40].…”

Vibrational relaxation as the driving force for wavelength conversion in the peridinin–chlorophyll a-protein

“…The system was coupled to a Nose-Hoover thermostat at 300 K [22]. The MD methodology was in most aspects identical to the one described in detail in a previous study [23].…”

Revealing Monoamine Oxidase B Catalytic Mechanisms by Means of the Quantum Chemical Cluster Approach

“…Furthermore, a conserved lysine residue (Lys296 in MAO B, Lys305 in MAO A) is located in a solvent-inaccessible hydrophobic area of the active site, establishing a well conserved Lys-water-N5 motif that is also found in other oxidases. 19 However, this conserved motif would be expected to fluctuate, with water molecules easily leaving and entering the active site, since the free energy cost linked to water displacement from and to the active site is small relative to the chemical step. In this situation, the lysine amino group should be subjected to strong electrostatic effects that might alter its acid/base equilibrium.…”